Our data reinforce the hypothesis that nutritional depletion is a key strategy for cancer treatment.Arginine is a semiessential amino acid required for the growth of melanoma and hepatocellular carcinoma, and the enzymatic removal of arginine by pegylated arginine deiminase (ADI) or arginase is being tested clinically. Here, we report a genetically engineered arginase FC fusion protein exhibiting a prolonged half-life and enhanced efficacy. The use of this enzyme to treat different tumor lines both inhibited cell proliferation and impaired cellular migration  Hepatocellular carcinoma (HCC) is the third most common cause of cancer death in the world, and the majority of patients with HCC are diagnosed at a late stage. Together with the lack of proven effective chemotherapy, the 5-year survival rate of HCC patients is only 7% worldwide . Amino acids serve as regulatory molecules that modulate numerous cellular functions and provide the substrates for protein synthesis. Therefore, amino acid deprivation is an effective treatment strategy for some cancers. Asparagine depletion by L-asparaginase is one of the best known examples, and it is widely used in clinical oncology for the treatment of acute leukemia and certain lymphomas . Seventy in vivo antitumor results in animals [Arginine deiminase (ADI) is a potent arginine-degrading enzyme from mycoplasma. Despite such concerns as immunogenic and toxic effects due to released ammonia , ADI was animals . As a popular and efficient way to improve the biopharmaceutical properties of bioactive proteins and peptides, pegylation has been widely used to ameliorate the stability, solubility, and immunological properties of bioactive compounds. However, precautions must be taken when using this method. Firstly, pegylation produces a population of drug conjugates. Secondly, these conjugates can accumulate in the liver, which ultimately leads to macromolecular syndrome. As an alternative method, biomolecules can be linked to the human Fc region of human immunoglobulin (IgG1), with demonstrated unique advantages. Numerous effector molecules, such as soluble cytokine receptors, retain their respective biological activities after coupling to the Fc domain of IgG1. The binding of Fc to FcRn receptors, which serves an important function in IgG homeostasis, results in increased half-lives of the fusion constructs. in vitro and in vivo for its antitumor activity. The results indicated that rhArg-Fc effectively inhibited the cellular growth of different tumors in culture and reduced tumor size in mice.Here, recombinant human arginase in the form of an Fc fusion protein (rhArg-Fc) was constructed and tested 6 Huh7 cells were removed from tissue culture plates using trypsin and injected s.c. into the back of the mice. The treatments were initiated after the size of each tumor reached approximately 5 mm in diameter. Six animals were used for each treatment group. The size of the tumors was monitored every 5 days for 50 days.Four-week-old athymic immunodeficient nude mice were obtained from Beijing Laboratory Animal Research Center. Approximately, 104 cells/well in 1 mL of culture medium and incubated for 24 h. The culture medium was replaced by a medium containing rhArg-Fc at different concentrations. After 3 days, the cellular growth and viability were measured using a tetrazolium salt assay [Cells were seeded in 24-well plates at a density of 2.5 × 10lt assay . The cellt assay . For theAmino acid analyses were performed using an Agilent HPLC, as described in . Brieflyi.v. with 100 μg/mouse and bled at 1, 2, 4, 6, 24, 72, and 144 h after injection. The levels of rhArg-Fc were measured with an ELISA assay using an antiarginase polyclonal antibody (Thermo Scientific) as the capturer. An antibody against the human Fc region (Jackson Immunology Research) was chosen as the reporter. Three CD1 mice were injected Human endothelial cells from umbilical cord vein were purchased from Allcells Inc. A confluent monolayer of synchronized HUVECS was scraped with a razor blade as described in . After w4 cells/well) using medium that had been pretreated with 0.5 IU/mL rhArg-Fc or not for 24 hr. Microvessel formation was observed using an inverted light microscope at 40x.Confluent HUVE cell monolayers were treated with 0.5 IU/mL rhArg-Fc or not for 48 h before being harvested and plated onto Matrigel-coated 24-well cluster plates . Cells were incubated for 3 h at 4°C and assayed by flow cytometry.Huh 7 cells were treated with rhArg-Fc at different concentrations for two days. Cells were washed with PBS, containing 1% BSA. Then, 3 mL of cold absolute ethanol was added, incubated at 4°C for 1 hr., washed, and provided with 1 mL of a 50 6 Huh7 cells were injected s.c. into the 4-week-old nude mice. The rhArg-Fc treatments were initiated after the size of the tumor reached approximately 5–10 mm in diameter. Six animals were used for each treatment group (cisplatin 0.5 mg/kg or rhArg-Fc 300 IU/mouse or a combination of both treatments). Mice were then treated twice a week for 2 weeks. The tumor size was monitored every 5 days for 50 days.10in vivo data, statistical analysis was done with SPSS. The differences in tumor sizes were determined by two-tailed Student's t-test.All data were based on at least three independent experiments. For the 2 and CH3 constant regions of the human IgG1 heavy chain. A vector directing the mammalian expression of rhArg-Fc was introduced into CHO cells to produce the secreted molecular protein. Taking advantage of the presence of the human IgG1 Fc region, we purified the fusion protein from the cell culture supernatant using a protein A column and eluted the column with acetate buffer (pH 3.8). We next examined the subunit structure of rhArg-Fc after SDS-polyacrylamide gel electrophoresis; the results showed that the subunit size is approximately 65 kd  and 1 melanoma cell line (SK-Mel-3) incubated with rhArg-Fc at multiple concentrations for 3 days. All of the tested human tumor cell lines were sensitive to the inhibition of both forms of rhArgs . The potive form . This reIn a separate assay, three days after rhArg-Fc treatment at the concentration of 0.2 IU/mL, rhArg-Fc was removed by intensive PBS wash. The addition of exogenous 1 mM L-arginine, but not L-glutamine, almost fully resumed cell growth . This fuTo further investigate the effect of rhArg-Fc on cell growth in more detail, we analyzed the effects of rhArg-Fc on the cell cycle distribution of Huh7. This was monitored by flow cytometry analysis after staining the cell DNA content with propidium iodide. In comparison with untreated controlled cells, 2-day treatment of rhArg-Fc at the concentration of 0.1 IU/mL resulted in an apparent increase of S-G2 phase cells. In addition, cells respond to rhArg-Fc in a dose-dependent manner as there are more S-phase arrested cells in the cells treated with 0.5 IU/mL of dosage. The S-phase fraction increased from 28% in controlled cells to 43% in 0.5 IU/mL treated cells .The depletion of arginine by ADI was recently reported to inhibit endothelial cell growth and migration; this antiangiogenic activity might be due to the suppression of nitric oxide (NO) generation , 16. To To further characterize the effect of rhArg-Fc on endothelial cell differentiation, we used a Matrigel assay to examine the effect of the fusion on microvessel formation, another key step in angiogenesis. Eighteen hours after treatment, the untreated endothelial cells differentiated into an elongated form and connected with each other to form networks. In contrast, rhArg-Fc significantly inhibited the differentiation of HUVEC cells into capillary-shaped tubes and resulted in sporadic vessel connection in a dose-dependent manner . in vivo stability of rhArg-Fc, the pharmacokinetics of rhArg-Fc was measured following the i.v. injection of mice. Blood samples were collected on different days, and the serum rhArg-Fc levels were tested by ELISA. The half-life of rhArg-Fc was approximately 4 days for the different sample groups treated with various amounts of rhArg-Fc . Indeed, the combined use of this concentration with rhArg-Fc at the same concentration led to a greater than 80% reduction in cell growth . No detectable arginine was observed one hour after incubation with rhArg-FC in vitro. Three days after treatment, rhArg-Fc inhibited cell growth in all of the 3 tested tumor cell types in a dose-dependent manner, with IC50 values ranging from 0.1 IU/mL to 0.3 IU/mL, which is comparable to its native form [Although arginase has been tested in experiments for many years , 25, limted form ; these asyndrome . An altesyndrome , 28. Thiive form . Our datWe further analyzed cell cycle and found that rhArg-FC significantly increased hepatoma cells in S-phase. This S-phase arrest may also result in the slightly elevated apoptosis represented as the sub-G1 cells shown in the FACS assay. Therefore, rhArg-Fc exerts its antitumor function though cytostatic (cell cycle arrest) and cytotoxic effects. Some other well-known antitumor agents such as cisplatin and irofulven also induce S-phase arrest to inhibit tumor proliferation . The mecWe found that rhArg-FC inhibits cell growth, cell migration, and capillary vessel formation in cultured HUVE cells. This inhibition of angiogenesis might be attributable to several reasons. Arginine is an essential amino acid for protein synthesis and polyamine formation, and depletion of arginine by arginase disrupts the nutrient supply for endothelial cell growth because polyamines are indispensable for endothelial cell proliferation . In addiin vitro cell culture, we found residual cells that survived during this period, and this group of cells was viable even when a high concentration of rhArg-Fc was used to deplete arginine from the medium. When HCC cells are maintained above the critical rhArg-Fc level, it is plausible that all of the arginine-sensitive clones are replaced by cells that are relatively resistant to arginine deprivation. This result is in agreement with our in vivo animal data and the observations of other groups [Although the majority of HCC cells were killed 3 days after the rhArg-Fc treatment in the r groups , 12, 26.Cisplatin is one of the most widely used drugs to treat various types of cancers. The antitumor activity of this drug is due to DNA crosslinking, interactions with cell surface nucleic acids, and the inhibition of methionine uptake into tumor cells , 33. At


In vitro, the proliferation, invasion and migration of U2-OS cells were suppressed by the inhibition of Aurora-B. These results suggest that Aurora-B may be involved in OS metastasis, and may be a promising target in the treatment of OS metastasis.Previous studies have suggested that Aurora-B may be involved in cancer metastasis. However, its role has been poorly evaluated in osteosarcoma (OS). The aim of this study was to investigate the correlation between Aurora-B expression and metastasis in human OS. The human OS cell line, U2-OS, and OS biopsy specimens were used in the study. The expression of Aurora-B protein was examined using immunohistochemistry and western blotting in OS tissues and U2-OS cells, respectively. AZD1152-hydroxyquinazoline-pyrazol-anilide, an inhibitor of Aurora-B, was used to inhibit Aurora-B expression in U2-OS cells. The effect of Aurora-B inhibition on U2-OS cell proliferation, invasion and migration was assessed using MTT, colony formation, wound healing and Transwell assays. The results showed that positive expression of the Aurora-B protein was observed in the nucleus, and that Aurora-B expression levels in the cases with pulmonary metastases were significantly higher than in those without metastasis.  Osteosarcoma (OS) is the most common tumor in bone and the third most common tumor in childhood and adolescence. Following the identification of effective chemotherapeutic agents, the five-year survival rates for patients treated with intensive multidrug chemotherapy and aggressive local control have been reported to be 55–80% –3. Howevet al , Aurora-B has been shown to be involved in a number of mitotic functions, including chromosome-microtubule interactions, sister chromatid cohesion, the spindle-assembly checkpoint and cytokinesis. Previous studies have shown that Aurora-B is upregulated in several types of human cancer, and that upregulation correlates with poor prognosis. As a result, Aurora-B has been suggested to be an important antitumor target –9. Li etet al  showed tet al . In addiet al –14. Howein vitro was investigated. AZD1152-hydroxyquinazoline-pyrazol-anilide (HQPA) was used to inhibit Aurora-B expression in U2-OS cells. Cell proliferation, migration and invasion were investigated using MTT, colony formation, wound healing and Transwell assays. The results revealed that there was a positive correlation between Aurora-B expression in OS tissues and pulmonary metastasis, and that cell proliferation, invasion and migration were inhibited by the inhibition of Aurora-B. The results indicated that Aurora-B may be involved in OS metastasis.In the present study, the expression of Aurora-B in OS with and without pulmonary metastasis was evaluated using immunohistochemistry (IHC). Furthermore, the effect of Aurora-B inhibition on cell proliferation, invasion and migration A total of 60 samples were obtained from patients with OS of the extremities who underwent surgery in The First Affiliated Hospital of Nanchang University . The examination of pulmonary metastasis was performed using plain films and chest computed tomography (CT) scans at initial diagnosis. None of the patients had a history of previous therapies with antitumor drugs or radiotherapy. There were 14 cases with pulmonary metastasis (23.3%), while 76.7% of cases were without metastasis. The samples were fixed with 10% formalin, embedded in paraffin and then cut into 4-μm sections. Informed consent was obtained from all participants, and the study protocol was approved by the Institutional Ethics Committee .Histological sections (4-μm) were stained with hematoxylin and eosin (H&E) and examined using IHC. IHC was performed using a streptavidin-peroxidase procedure. Briefly, antigen retrieval was performed by heating the deparaffinized, rehydrated sections in 10 mM citrate buffer (pH 6.0) for 20 min, followed by blocking with 10% goat serum. The sections were then incubated overnight at 4°C with the primary antibody  at a final dilution of 1:500. For the negative controls, the sections were incubated with phosphate-buffered saline (PBS) instead of antibodies. After being washed three times with PBS, the sections were incubated with biotinylated secondary antibody for 40 min and then incubated with horseradish peroxidase (HRP)-conjugated streptavidin for 30 min. The sections were subsequently subjected to chemiluminescent staining and counterstained using hematoxylin. The stained sections were evaluated and scored by two doctors of pathology, in a blind manner and without prior knowledge of the clinical pathological features of the patients. The expression levels of Aurora-B were judged according to staining intensity, following the examination of ≥500 cells in five representative areas, and the intensity scores were recorded as follows: None, 0; weak, 1; moderate, 2; and intense, 3. According to the percentage of tumor cells that were positive for Aurora-B expression, the following percentage scores were assigned: 0% (score 0); >10% (score 1), 11–50% (score 2), 51–80% (score 3), and 81–100% (score 4). The final score was averaged with the scores from the two doctors of pathology; these scores were calculated by adding the intensity score to the percentage score. A final score of <4 was defined as (−), while scores of 4 and 5 were defined as (+) and (++), respectively, and a score of ≥6 was defined as (+++).2.The U2-OS human OS cell line was obtained from the American Type Culture Collection , and the cells were routinely cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  in a humidified 37°C incubator containing 5% COU2-OS cells were cultured in 96-well tissue culture plates at a cell density of 5,000 cells per well in Minimum Essential Media  containing 10% FBS and 2 mM L-glutamine. Following attachment overnight, the medium was replaced and the cells were incubated with increasing concentrations  of AZD1152-HQPA for 24, 48 and 72 h. Subsequently, MTT assays were performed in triplicate at a wavelength of 490 nm.6/ml/well) were seeded in tissue culture plastic dishes and treated with AZD1152-HQPA (100 nM) for two weeks to form colonies. The formed colonies were stained with Giemsa, and the colonies containing >50 cells were counted under an inverted microscope . Six independent experiments were performed over multiple days.U2-OS cells  for 24 h. Total protein from the cells was extracted using radioimmunoprecipitation assay lysis buffer containing 60 μg/ml phenylmethylsulfonyl fluoride. Protein concentrations were assessed using a bicinchoninic acid protein assay kit . The protein samples were denatured at 100°C for 10 min and then preserved at −20°C for later use. The proteins were separated by SDS-PAGE and transblotted onto polyvinylidene difluoride membranes. The membranes were then probed with rabbit anti-Aurora-B monoclonal antibody  or β-actin antibody  overnight at 4°C. Following incubation with the appropriate anti-rabbit or anti-mouse HRP-conjugated secondary antibody  for 1.5 h at room temperature, immunoreactive bands were visualized using chemiluminescence dissolvent  and exposed to X-ray film . The assessment of the grayscale values was performed using ImageJ . All experiments were repeated six times over multiple days.The invasion of U2-OS cells was measured using the BD BioCoat™ BD Matrigel™ Invasion Chamber  in accordance with the manufacturer’s instructions. The medium in the lower chamber contained 5% FBS as a source of chemoattractants. Cells were suspended in serum-free medium containing 100 nM AZD1152-HQPA and added to the upper chambers at the same time. The cultures were rinsed with PBS and the medium was replaced with fresh medium alone or medium supplemented with 10% FBS. The cells were then incubated at 37°C for 24 h. Cells that passed through the Matrigel-coated membrane were stained with Diff-Quik  and photographed. Cell migration was quantified using direct microscopic visualization and counting. The values for invasion were obtained by counting three fields per membrane and represented the average of six independent experiments performed over multiple days.in vitro ‘wound healing assay’. In brief, cells were grown to confluence in six-well tissue culture plastic dishes to a density of ~5×106 cells/well. Following treatment with 100 nM AZD1152-HQPA for 24 h, the cells were denuded by dragging a rubber policeman  through the center of the plate. Cultures were rinsed with PBS and the medium was replaced with fresh medium alone or medium containing 10% FBS. The cells were then incubated at 37°C for 24 h. Photographs were taken at 0 and 24 h, and the migration distance was measured. The cell migration rate was obtained by counting three fields per area and represented the average of six independent experiments performed over multiple days.Cell migration was assessed by examining the ability of the cells to move into a cellular space in a two-dimensional All measurement data are presented as the mean ± standard deviation. Statistical analysis was performed using the independent-samples t-test, and the two-independent-samples test was used for the analysis of the correlation between Aurora-B protein expression levels and pulmonary metastasis. P<0.05 was considered to indicate a statistically significant difference. All analyses were performed using SPSS statistical software version 13.0 .Aurora-B was expressed in the nucleus , and thein vitro, which suggested that Aurora-B may be a promising target for the treatment of OS.In order to investigate the effect of Aurora-B inhibition on U2-OS cell growth, AZD1152-HQPA, a specific inhibitor of Aurora-B, was used to suppress Aurora-B expression in the U2-OS cells. The cells were treated with various concentrations  of AZD1152-HQPA, and MTT assays were performed to measure the inhibitory effect of AZD1152-HQPA on U2-OS cells proliferation. The results of the MTT assays revealed that AZD1152-HQPA inhibited U2-OS cell proliferation in a dose- and time-dependent manner . The IC5in vitro.According to the IC50 value, the appropriate concentration of AZD1152-HQPA for wound healing migration and Transwell invasion assays was determined. To examine the effect of Aurora-B inhibition on the mobility of U2-OS cells, the migration and invasion were measured using wound-healing and Transwell assays, respectively. The cells were treated with 100 nM AZD1152-HQPA for 24 h. In the Transwell invasion assays, the invasion of the cells treated with AZD1152-HQPA was significantly inhibited when compared with that of the untreated cells , and these kinases are expressed at maximum levels during mitosis. Aurora-B, part of the CPC, is located on the chromosome arms during prophase and at the centromeres during prometaphase and metaphase. The kinase subsequently localizes to the midbody during cytokinesis. Aurora-B has been shown to be overexpressed in a number of types of cancer ,15–17 Inin vitro investigations. Preliminary studies showed that AZD1152 was active against several types of solid tumors, including colon, breast and lung cancers (in vitro.Recent studies revealed that Aurora-B inhibition was capable of blocking cell proliferation and inducing cell apoptosis in several types of tumor ,19. Thes cancers ,26. Howein vitro.Notably, studies recently showed that the upregulated expression of Aurora-B was associated with tumor cell metastasis, and that the downregulation of Aurora-B was capable of inhibiting cell invasion and migration in various types of tumors ,14,27,28in vivo are required to elucidate the potential of Aurora-B as a target for the treatment of OS metastases and a predictor of prognosis.In conclusion, this study indicated that Aurora-B may be involved in the development, progression and metastasis of OS, and that targeting Aurora-B may be a potential treatment strategy for OS management. However, in the present study the number of OS tissues was low. Furthermore, the tumor microenvironment is important in tumor development, progression and metastasis and therefore, further experiments


Thermococcus profundus) was further immobilized by physical adsorption. The modified electrode (GC/MWCNTs/L-proDH) exhibited an electrocatalytic signal for L-proline compared to bare GC, GC/L-proDH and GC/MWCNTs electrodes, which suggested that the presence of MWCNTs efficiently enhances electron transfer between the active site of enzyme and electrode surface. The immobilized L-proDH showed a typical Michaelis–Menten catalytic response with lower apparent constant.A glassy carbon electrode (GC) was modified by multi-walled carbon nanotubes (MWCNTs). The modified electrode showed a pair of redox peaks that resulted from the oxygen-containing functional groups on the nanotube surface. A recombinant thermostable dye-linked L-proline dehydrogenase (L-proDH) from hyperthermophilic archaeon ( As an essential amino acid for the proper functioning of tendons and joints in the human body, the quick and sensitive determination of L-proline is of importance for both pharmaceutical and food industries . ClassicIn recent years, researchers have paid much more attention to the construction of electrochemical enzyme biosensors for the analysis of amino acids –7, and sIn the last decade, the use of nano materials, especially carbon nanotubes (CNTs), in the construction of enzyme biosensors has received considerable attention. Because of their excellent mechanical and electrochemical properties –14, CNTsThermococcus profundus) was used to fabricate L-proline sensor based electrodes modified by layer-by-layer self-assembly immobilization of dye-linked L-proDH and ferrocene-labeled redox polyelectrolytes [In our previous work, the recombinant thermostable dye-linked L-proline dehydrogenase (L-proDH) from hyperthermophilic archaeon 6 as the probe. Typical reversible cyclic voltammograms were observed for both the bare and MWCNTs modified electrodes (data not shown here). It was clearly demonstrated that the current obtained on the GC/MWCNTs was nearly 2.5 times greater than that with a bare GC. The larger current response indicated an increased electrode surface or a fast electron process after modification by MWCNTs. The cyclic voltammograms of the GC/MWCNTs electrode in phosphate buffer  at different scan rates (from 5 to 200 mV s−1) are displayed in E0’=(Epa+Epc)/2) of approximately +0.02 V. In the insert figure of The electrochemical properties of GC/MWCNTs were first investigated by cyclic voltammetry with KE0’ and buffer pH was found, with the linear regression equation of E0’(V) = 0.4001−0.0508 pH . Similar results were found by other groups [The cyclic voltammograms of the GC/MWCNTs electrode in phosphate buffer at different pHs were also studied . The redr groups –30 and ir groups .−1 and 1075 cm−1 are due to a carbonyl group and C–O bond, respectively. This also indicates the existence of oxygen-containing groups on nanotube surface. As commercial MWCNTs were used in this research, some oxygen-containing groups must have been formed after preparation. Zhang et al. also found these kinds of functional groups when freshly prepared nanotubes were exposed to air after one day, and pointed out that the redox waves of these groups cannot be eliminated by removal of the dissolved oxygen in buffer [In the IR spectrum of the MWCNTs sample shown in n buffer .+ + e− = =COH.The influence of scan rate on the anodic and cathodic peak positions is shown in Dye-linked L-proDH was further immobilized on a MWCNTs-modified electrode, and its electrochemical behavior was studied. Typical cyclic voltammograms of GC, GC/L-proDH, GC/MWCNTs and GC/MWCNTs/L-proDH electrodes in the absence and the presence of L-proline are shown in L-proline and I is displayed in the insert figure of The amperometric response of the GC/MWCNTs/L-proDH electrode to L-proline was further investigated at an applied potential of +0.2 V. The current response increased with successive additions of substrate and with stirring as shown in −2 for 1 mM L-proline with a detection limit of 0.05 mM (S/N = 3), which was much higher than that obtained in previous work which used a ferrocene-labeled redox polymer as the electron mediator [The amperometric response was about 0.87 μA cmmediator ,28, and L-proline is plotted in L-proline was clearly found, which is consistent with the following Lineweaver–Burk equation.The Lineweaver–Burk curve of 1/I vs 1/CMapp) of the immobilized L-proDH on MWCNTs-modified electrode was calculated to be 0.33 mM, which was lower than that of free L-proDH (2.05 mM) in buffer with DCPIP as the electron acceptor [According to the slope and intercept of Lineweaver–Burk equation, the apparent Michaelis–Menten constant (KMapp) of the immobilized L-proDH on MWCNTs-modified electrode was lower than that of the free enzyme in buffer solution with DCPIP as the electron acceptor.In this work, both MWCNTs and dye-linked L-proDH were immobilized on an electrode surface, and the electrochemical behavior of L-proDH was investigated. On the MWCNTs-modified electrode, a pair of broad redox peaks was observed, which were shifted to more positive values with decreasing pH. These peaks were probably due to the oxygen-containing functional groups on nanotube surface. Based on these oxygen-containing functional groups, electrical communication between the FAD active sites of L-proDH and electrode were set up, and the electrode immobilized with both MWCNTs and L-proDH exhibited a catalytic current response to L-proline at a relatively lower potential. The GC/MWCNTs/L-proDH electrode showed a typical Michaelis–Menten response to the addition of L-proline in amperometric measurements, and the apparent Michaelis–Menten constant  with a diameter of 10–20 nm and length of 5–15 μm were purchased from Tokyo Chemical In.  and used as received. L-Proline and 2,6-dichlorophenolindophenol (DCPIP) were obtained from Wako Chemical In. , and glutaraldehyde . All the other chemicals were of analytical grade.An FT-IR spectrometer  was used to characterize the MWCNTs sample. The IR pellet was made by mixing a small amount powdered KBr and the nanotube sample, and the IR spectra was recorded. A field emission scanning electron microscopy  was used to observe the surface of MWCNTs-modified electrode. All electrochemical experiments were performed on a potentiostat  connected to a personal computer. A typical three-electrode system was used, with a 3 mm diameter of glassy carbon electrode (GC) as the working electrode, a platinum wire as the counter electrode and Ag/AgCl (3 M NaCl) as the reference electrode. The GC electrodes were finely polished by 1.0 μm and 0.05 μm alumina, and cleaned by ultrasonication in Milli-Q water for 30 s before modification.4 M−1 cm−1 was used to calculate the reduction of DCPIP, and one unit of activity was defined as the amount of enzyme that reduced one μmol of DCPIP per min at 50 °C. The protein concentration of the L-proDH solution was found to be 1.6 mg mL−1 based on the classical Bradford method, and the specific activity of enzyme was calculated to be 1.8 units mg−1.Recombinant dye-linked L-proDH, from hyperthermophilic archaeon, was prepared and purified according to the literature method . The enzA MWCNTs solution was prepared by dispersing 1.0 mg of MWCNTs in 1.0 mL ethanol followed by ultrasonication for 30 min. A 10 μL aliquot of the MWCNTs dispersion solution was dropped onto the top of the pre-treated GC electrode, which was dried at room temperature for 2 h and is referred to as GC/MWCNTs.2 stream at room temperature. The electrode with the immobilized enzyme was washed three times with Milli-Q water, and stored in phosphate buffer  at 4 °C when not in use. The electrode modified with both MWCNTs and L-proDH is referred to as GC/MWCNTs/L-proDH. In controlled experiments, L-proDH was immobilized on a bare GC electrode surface by a cross-linking method using glutaraldehyde as the linking agent [2 stream at room temperature. The electrode so obtained is referred to as GC/L-proDH.L-proDH was immobilized on MWCNTs-modified GC electrode by physical adsorption. A 10 μL L-proDH solution was dropped onto a MWCNTs-modified electrode, and dried in a Nng agent –36. A 302 for 15 min before measurements to eliminate the influence of dissolved oxygen. The amperometric detection was performed at +0.2 V vs Ag/AgCl, and the substrate was added after the current reached the baseline.All electrochemical measurements were carried out in 0.3 M phosphate buffer (pH 7.5) at room temperature (20 °C). The buffer solution was purged with high-purity N


EAR8-II was designed to inherit advantages from two previously introduced peptides, AAP8 and EAK16-II, in their self/co-assembly. This peptide is short, simple, and inexpensive to synthesize, while possessing a low critical assembly concentration (CAC). The choice of alanine (A) residues in the peptide sequence provides moderate hydrophobic interactions, causing a minimal degree of aggregation, compared with other more hydrophobic residues. EAR8-II is an ionic-complementary peptide, similar to EAK16-II, can self/co-assemble with hydrophobic compounds such as THP, and forms a stable fibular nanostructure in aqueous solution. Physiochemical properties and cellular activities of the EAR8-II and THP complexes were evaluated and show dependency on the peptide-to-drug ratio. The complex at the peptide-to-drug mass ratio of 5:1 provides a stable solution, uniform nanostructure, and highly effective anticancer activity against various cancer cell lines. This work forms the basis for detailed studies on EAR8-II and THP formulations  Despite its practicability in anticancer drug encapsulation, AAP8 shows uncontrollable aggregation, which is not favorable for many biological applications. Therefore, a DEG-functionalization procedure was explored as a strategy to improve the multi-functionality of the nanocarrier by avoiding aggregation and to enhance bioavailability of the drug delivery system  = 0.3 mg/mL, followed by stabilization of the particles in the smaller range of hydrodynamic diameter ~500–700 nm, where at higher [EAR8-II] = 0.8–1 mg/mL, the solution become unstable with larger particles . At low +] at higher peptide concentrations. Since THP has a pKa of ~7.3 (pyridine-like nitrogen) it can be protonated in a weakly acidic environment. Consequently, acidic EAR8-II produced protonation of THP at higher peptide-to-drug ratios and led to more positive zeta potential values . Cellular viability profiles for both HeLa and A549 cells at different ratios followed a similar trend, where the cell viability of A549 was relatively higher than that of HeLa, indicating more sensitivity of HeLa cells toward EAR8-II-THP complexes. In the absence of peptide in the complex, THP has been shown to induce non-significant cellular toxicity against both cell lines. Whereas, when EAR8-II stabilized THP in aqueous solution, cellular toxicity of both cells increased significantly. Due to the instability of the THP molecules in aqueous solution they do not penetrate the cell membrane effectively.As shown in It is speculated that protonated THP molecules were inclined to interact with negatively charged cell membrane and promoted cellular uptake. The hydrophobic properties of THP further stimulated molecules cross the cell membrane and localized in the cytoplasm. As shown in A similar pattern was observed in the fluorescence microscopy images from the cells treated with EAR8-II-THP complexes at different ratios. For both cell lines, complexes with peptide concentrations of 0.3 and 0.5 mg/mL resulted in the highest cellular uptake of THP in cytoplasm. In contrast, complexes at lower and higher concentrations of the peptide showed lower localization of THP in cytoplasm. Note that the bright red fluorescence observed in 1:2 and 1:1 ratio complexes were from non-dissolved THP not localized in cytoplasm. At the ratios of 3:1 and 5:1, the localization of THP in cytoplasm was very uniform, whereas other ratio complexes had THP randomly localized or just accumulated on the membrane . These ret al. emphasized the importance of drug stability upon dilution during treatment time in clinical trials  concentrations at 0.1 mg/mL (0.159 mM), followed by mechanical stirring at 900 rpm for 24 h.3.2.2.i.e., the relationship between the size of a particle and its scattered light intensity obtained with the multimodal Contin algorithm, which was provided in the Dispersion Technology Software 5.1 package .The particle size distribution and zeta potential of peptide-drug complexes at different peptide-to-drug ratios were measured by a Zetasizer Nano ZS . Appropriate settings, viscosity, refractive index, and dispersant solvent were set for each measurement at 25 °C. A small 50 μL volume of the sample was transferred from the vial to a disposable solvent resistant micro cuvette . The scattered light intensities of the samples were collected at an angle of 173°. This was known as backscatter detection, For zeta potential measurements, the sample was injected into a 1 cm × 1 cm disposable cuvette with a volume of ~1 mL, and a dip cell (ZEN1002) was inserted into the solution so that the solution covered the electrodes on the dip cell. The zeta potential distribution was directly calculated from electrophoretic mobility distribution based on the Smoluchowski formula. For each solution, the zeta potential was repeated three times. The values reported herein correspond to an average of the peak values of the zeta potential distribution from replicates.3.3.3.The THP fluorescence emission spectra were acquired on a photon technology international spectrafluorometer  with a continuous xenon lamp as the light source. The emission spectra were collected in the wavelength range of 500–800 nm, while samples were excited at 480 nm. The maximum emission peak of THP is at a wavelength of 590–600 nm. The excitation spectrum was collected in the wavelength range of 200–500 nm. The maximum excitation peak of THP is at wavelength of 470–490 nm. The excitation and emission slit widths of the monochromators, which control the amount of light coming in and out of the sample chamber, were set at ½ and 2 turns to yield a spectral resolution of 1 and 4 nm, respectively. The fluorescence intensity at 590 and 480 nm for emission and excitation were obtained by taking the average of twenty points of fluorescence intensity, respectively. Each fluorescence spectrum was divided by lamp intensity to account for eventual lamp fluctuations . The nor3.3.4.4 cells per well concentrations, were seeded in 96-well plates, followed by treatment with different peptide-drug complex ratios for 24 h. Cellular-toxicity experiments were performed with sensitive colorimetric assay called Cell counting kit-8 (CCK-8) . CCK-8 contains highly water-soluble tetrazolium salt (WST-8), which is reduced by dehydrogenase activities in cells to give the yellow color of the formazan dye, which is soluble in culture media. The amount of formazan dye is measured by absorption (O.D.) at 450 nm, which is directly proportional to the number of living cells.Two human cancer cell lines, with 105 cells per well. They were then treated with peptide-drug complexes for 2–4 h. For spectroscopy with the PTI system, after 6 h incubation at 37 °C, the cells were lysed with 1% SDS in Tris-EDTA, followed by probe sonicating to complete cell lysis. The lysed cell suspensions were excited at 480 nm, and emission spectra were collected from 500 to 800 nm to quantify the THP content of the treated cells. Each spectrum was corrected by spectra of lysed untreated cells. The amount of drug uptake is directly proportional to the emission peak intensities. Higher fluorescence emission intensity indicates higher cellular uptake of THP.Cellular uptake of THP was also carried out by spectroscopy, exploiting the intrinsic fluorescence properties of THP . Cells were plated in 12-well plates, with concentrations of 2 × 104.50 of THP was calculated as ~19.3 μM for A549 >~1.8 μM for HeLa cells, indicating sensitivity of HeLa cells compared with A549 toward EAR8-II-THP complexes. This study provides a platform for using self/co-assembly peptides as carriers for hydrophobic drugs in future for in vitro and in vivo applications by introducing a stable complex with relatively high therapeutic efficacy against cancer cells.The current article introduced a new self/co-assembling ionic-complementary peptide, EAR8-II, stabilizing the hydrophobic drug pirarubicin (THP) in protonated form in aqueous environments, as shown by surface charge and fluorescence measurements. Results show the complex formation to be peptide-to-drug ratio dependent. Increasing EAR8-II concentration at a constant THP concentration reduced the pH value of the complex and consequently caused protonation of THP stabilized in solution. At lower peptide-to-drug ratios, an insubstantial amount of THP molecules were solubilized in aqueous solution, according lower fluorescence emission intensity than in complexes with higher peptide-to-drug ratios, in which more THP is stabilized. The peptide-to-drug ratio affected the particle size distribution within a complex; where at very low and very high concentrations of EAR8-II, the average particle size was large, indicating the presence of long fibers and large aggregates in the solution. However, in complexes with peptide concentrations between 0.2 and 0.6 mg/mL, the average particle size observed was ~600 nm. Cytotoxicity and cellular uptake by cancer cell lines demonstrated peptide-to-drug ratio dependent behavior, in which, at very low and very high ratios, the anticancer activity was lower than that of peptide-to-drug mass ratios between 2:1 and 5:1. Deficiency and excess of the peptide in stabilizing THP caused not fully encapsulated and highly trapped THP molecules, respectively. The complex at a 5:1 peptide-to-drug mass ratio was recognized as an optimum peptide-to-drug ratio complex where the highest encapsulation efficiency was observed for the lowest peptide concentration present in the complex. This complex also offered the highest anticancer activity and cellular uptake against cancer cells compared to other ratio complexes. The anticancer activity of this complex and its serial diluted solutions were further studied against two cancer cells. The IC


Gastric cancer (GC) is one of the most common malignancy and primary cause of death in Chinese cancer patients. Recurrence is a major factor leading to treatment failure and low level of 5-year survival rate in GC patients following surgical resection. Therefore, identification of biomarkers with potential in predicting recurrence risk is the key problem of the prognosis in GC patients.A total of 74 GC patients were selected for systematic analysis, consisting of 31 patients with recurrence and 43 patients without recurrence. Firstly, miRNAs microarray and bioinformatics methods were used to characterize differential expressed miRNAs from primary tumor samples. Following, we used a ROC method to select signature with best sensitivity and specificity. Finally, we validated the signature in GC samples (frozen fresh and blood samples) using quantitative PCR.P<0.001). In addition, we evaluated that hsa-miR-335 were involved in regulating target genes in several oncogenic signal-pathways, such as p53, MAPK, TGF-β, Wnt, ERbB, mTOR, Toll-like receptor and focal adhesion.We have identified 12 differential miRNAs including 7 up-regulated and 5 down-regulated miRNAs in recurrence group. Using ROC method, we further ascertained hsa-miR-335 as a signature to recognize recurrence and non-recurrence cases in the training samples. Moreover, we validated this signature using quantitative PCR method in 64 test samples with consistent result with training set. A high frequency recurrence and poor survival were observed in GC cases with high level of hsa-miR-335 (Our results indicate that the hsa-miR-335 has the potential to recognize the recurrence risk and relate to the prognosis of GC patients. Currently, surgery is still the main option for treating gastric cancer (GC), however even after performing curative resection, approximately 40–65% of GC patients will experience a recurrence of the disease possibly encompassing local relapse, peritoneal dissemination or hematogenous metastasis Several risk factors, including pathological serosa state, margin status, tumor microvessel density and gene expression changes related with recurrence have been reported A recently discovered of miRNAs are highly conserved in the genomes of invertebrates, vertebrates and plants. They serve critical functions in multiple biological processes miRNAs have been presented as effective potential biomarkers for tracing the tissue origin in malignant tumors of unknown primary origin In this study, we have identified a miRNA (hsa-miR-335) having potential to predict recurrence risk of GC based on microarray and clinical data of a small-size sample in Chinese GC patients. Our results showed that hsa-miR-335 could be act as a clinical prognostic signature for GC.This study has been approved by the Ethics Committee of Wuhan General Hospital of Guangzhou Command, PLA. All gastric cancer patients and non-cancer patients with digestive diseases provided written informed consent in our study.Patients undergoing gastrectomy for potentially curable GC at the Wuhan general hospital of Guangzhou military command were subjects in this study. Eligibility for inclusion in this study included histologically confirmed adenocarcinoma of the stomach or gastroesophageal junction. All patients had received complete resections including an attempt at complete tumor removal with inclusion of wide negative margins and extended retroperitoneal lymphadenectomy (D2 type). Information on clinicopathologic, therapeutic, and outcome parameters of patients from May 2005 to Feb 2012 was collected retrospectively. Cancer staging was performed according to the fifth edition of the American Joint Commission on Cancer TNM criteria.Recurrence was defined as any cancer recurrence including lymph node, remnant stomach, local, peritoneal and hematogenous metastases. All patients that experienced recurrence of cancer were diagnosed clinically or radiographically, and confirmed by biopsy via upper gastrointestinal endoscope or percutaneous puncture. The radiographic standard for the recurrence diagnosis included CT or MRI of the chest, abdomen, pelvis, head and bone scans, or other diagnostic tests which were used only under special circumstances. All of the samples were obtained from surgical specimens of patients with gastric adenocarcinoma and all patients gave written consent for the use of these tissues for research purposes. We selected samples (including frozen fresh and blood samples) from 74 patients with and without GC recurrence.http://www.capitalbio.com) and also according to Hua’s procedure The miRNA microarray analysis was performed as described in detail on the website of CapitalBio . For blood samples, miRNA were extracted using the miRNease kit (Qiagen). In brief, 700 µl of QIAzol reagent was added to 200 µl of plasma sample and incubated for 5 min at room temperature. Then, 140 µl of chloroform was added, vortexed for 15 sec and incubated for 3 min at room temperature. This was followed by a centrifugation of 14000 rpm at 4°C for 15 min. The upper, watery phase was removed and 1.5 times of its volume added in 100% ethanol. Each 700 µl of mixture were placed on a column and centrifuged at 13000 rpm at room temperature for 15 sec. Following, 500 µl Buffer RPL was added to the column and centrifuged at 13000 rpm at room temperature for 2 min. The column was then centrifuged at 13000 rpm at room temperature to dry it. The following was the elution step with 20 µl of nuclease-free water. The eluted RNA was stored at −70°C.http://microrna.sanger.ac.uk/sequences/). Stem-loop reverse transcription (RT) primers were designed according to Chen C−ΔΔT formula and normalized to U6 snRNA expression Mature miRNA sequences were acquired from the Sanger Institute miRBase Sequence Database . The AUC curves provided a measure of the overall performance of a diagnostic test. The ratio of miRNA signal intensities and Ct value of each miRNA were used for ROC calculation in samples. Survival analyses were also performed by the MedCalc software package.P<0.05 was considered statistically significant.The clinical data were analyzed using the Chi-Square test. The cumulative survival curve was compared by the log-rank test. For all analyses, a difference with http://www.targetscan.org) to select plausible targets of the differential expressed miRNAs. An integrated gene ontology database molecular annotation system  was used to investigate the miRNA targeted genes and their involvement in various signal pathways.We utilized a miRNA target gene prediction database TargetScan , Age (P = 0.502), Tumor location (P = 0.299), Differentiation (P = 0.511), Lymph node resection (P = 0.217) and Status of adjuvant chemotherapy (P = 0.214). There were significant difference in UICC stage (P = 0.108) and survival/death ratio noted , with median survival time of 18.9 months in recurrence vs. 43.9 months in non-recurrence group respectively (P<0.001) (There were no significant difference in the Sex (P<0.001) .We used a miRNAs microarray chip to measure miRNAs expression levels in 10 GC samples with/without recurrence (5/5) as training set. When comparing miRNA expression between the groups, 7 up-regulated and 5 down-regulated miRNAs were found that were statistically significant in the GC recurrent group . These 1 hsa-miR-335 = 4.675; FC hsa-miR-128b = 1.453) of each biomarker, we select hsa-miR-335 for further study.We used ROC method to analyze the sensitivity and specificity of the candidate biomarkers based on miRNA microarray data. We obtained 2 miRNAs hsa-miR-335 and hsa-miR-128b with best sensitivity and specificity in classifying GC samples with and without recurrence , Table 3The expression level of hsa-miR-335 was detected by real-time PCR in 64 test GC samples compared with the matched adjacent tissue as control. The results showed that in 26 cases with recurrence, 21 cases  were high-expressed of hsa-miR-335; and in 38 cases without recurrence, 29 cases  were low-expressed of hsa-miR-335 . The samIn addition, we detected the expression level of hsa-miR-128b in 64 GC blood samples using real-time PCR. The results showed that in 26 cases with recurrence, 18 cases  were high-expressed of hsa-miR-128b; and in 38 cases without recurrence, 27 cases  were low-expressed of hsa-miR-128b. The FC value of hsa-miR-128b and hsa-miR-335 were 1.647 and 3.589 when compared recurrence with non-recurrence samples, respectively . We alsoP<0.001; P<0.001; The 74 GC patients were divided into two groups, including 31 patients with recurrence as a group and 43 patients without recurrence as a group. The patients who experienced a recurrence of GC had a significantly reduced median survival rate P<0.001; . We deteIn order to investigate the possible regulation mechanisms of the hsa-miR-335 in the process of GC recurrence, we utilized a bioinformatics database to select plausible targets of this miRNA. A total of 255 genes were predicted as the target genes of hsa-miR-335. Signaling pathway analyses showed that most of the targeted genes which were regulated by hsa-miR-335 were involved in the same pathways, such as p53,MAPK, TGF-β, Wnt, ERbB, mTOR, Toll-like receptor, focal adhesion . We propRecurrence is frequent in GC patients following surgery; therefore it is important to identify cases with a high recurrence risk. Traditional clinic pathological factors are sometimes inadequate for prediction of recurrence in individuals and many research groups have attempted to identify biomarkers using new technologies that might distinguish these high-risk cases. A number of recent investigations have documented miRNA alterations that are involved in the initiation and progression of human cancers. miRNA expression profiling of human tumors have identified expression signatures associated with diagnosis, staging, progression, prognosis and response to treatment.In this study, we analyzed primary GC cases in order to predict disease recurrence and defined non-recurrent cases as those free of GC for at least one year after curative resection. We identified hsa-miR-335 as a plausible prognosis signature of GC based on the microarray data from 10 GC samples as training set. Furthermore, we validated this signature on 64 test samples with more than an average of 85% sensitivity and specificity. The survival analyses results showed that patients with high expression levels of hsa-miR-335 had reduced overall survival rate compared to those with low expression levels of hsa-miR-335. These results indicate that this signature had the potential to predict recurrence of GC.Microarray technology has developed significantly and become a comprehensive and useful method to help us better understands cancers Until now, the miRNA designated in this study to predict the risk of GC recurrence have not been well-characterized. hsa-miR-335, which is transcribed from the genomic region chromosome 7q32.2, has been reported to differential expressed in benign and malignant tumours Blood is the most accessible sample in hospitals. The clinical significance of identification of biomarkers which could be easy detected just as hsa-miR-335 is obviously. In addition, archived paraffin-embedded specimens are also one of the readily available materials in hospitals, which are often kept with well documented clinicopathological data. They represent excellent sources for prognostic application in both basic research and clinical studies. miRNAs range in size from 19–25 nt and they are protected by the RNA-induced silencing complex, which may render them less susceptible to RNA degradation compared to mRNA in these tissues In summary, we have identified a prognosis-related miRNA which can predict recurrence risk of GC patients. By combining this signature with conventional clinicopathologic factors, we should be able to predict a patient’s disease outcome more accurately. Additionally the identification of high-risk patients would lead to the consideration of additional therapeutic intervention and may thus inform the physician of a better follow-up program.


The MODerate resolution Imaging Spectroradiometer (MODIS) and the Advanced Spaceborne Thermal Emission Reflection Radiometer (ASTER) are onboard the same satellite platform NASA TERRA. Both MODIS and ASTER offer routine retrieval of land surface temperatures (LSTs), and the ASTER- and MODIS-retrieved LST products have been used worldwide. Because a large fraction of the earth surface consists of mountainous areas, variations in elevation, terrain slope and aspect angles can cause biases in the retrieved LSTs. However, terrain-induced effects are generally neglected in most satellite retrievals, which may generate discrepancy between ASTER and MODIS LSTs. In this paper, we reported the terrain effects on the LST discrepancy with a case examination over a relief area at the Loess Plateau of China. Results showed that the terrain-induced effects were not major, but nevertheless important for the total LST discrepancy. A large local slope did not necessarily lead to a large LST discrepancy. The angle of emitted radiance was more important than the angle of local slope in generating the LST discrepancy. Specifically, the conventional terrain correction may be unsuitable for densely vegetated areas. The distribution of ASTER-to-MODIS emissivity suggested that the terrain correction was included in the generalized split window (GSW) based approach used to rectify MODIS LSTs. Further study should include the classification-induced uncertainty in emissivity for reliable use of satellite-retrieved LSTs over relief areas. It was launched on December 18, 1999 and began collecting data on February 24, 2000 . Being oEither ASTER or MODIS provides data are used for routine retrieval of land surface temperatures (LSTs). ASTER LST has a spatial resolution of 90-m with coverage of 60-km by 60-km. The temperature and emissivity separation (TES) algorithm allows accurate retrieval of LST products using the ASTER multi-spectral thermal infrared (TIR) bands . MODIS Let al. compared ASTER and MODIS LSTs over a semi-arid and a Savannah area, and found no significant ASTER-to-MODIS LST differences caused by spatial heterogeneity [et al. showed that spatial heterogeneity made the effects negligible [et al. developed a correction approach to rectify the underestimated MODIS 1-km LST product [et al. used the Wan et al.'s approach to reduce the LST discrepancy between ASTER-to-MODIS [et al.'s approach using the Planck function and ASTER emissivity data. In addition, Liu et al. proposed another correction approach based on the principle of the GSW algorithm. So far, all the correction techniques showed the improved agreement between ASTER and MODIS LST products [Although simultaneous observation eliminates the differences in ASTER and MODIS LST data due to time differences, recent studies have reported a discrepancy in ASTER and MODIS LSTs by approximately 3 K over semi-arid areas ,11,12. Togeneity . Using a product . Recentlet al. found that terrain effects contributed approximate 0.7 K to the ASTER-to-MODIS discrepancy in a rugged area on the Loess Plateau of China [Both ASTER and MODIS LST products have been applied worldwide. It is well-known that a large fraction of the earth surface consists of mountainous areas. Variations in elevation, terrain slope and aspect angles can interact with satellite viewing geometry to cause biases in retrieved LSTs . Therefoof China . Howeveret al. [In this study, we use the data sets same as Liu et al.  to analy2.In the study, we adopt three approaches developed in ,12. One 2.1.T is the corrected LST and T′ the satellite-derived LST. γ is the angle between the satellite-view path and the normal to the terrain element. To a thermal band, the angle of emitted radiance can be geometrically determined from the following equation:α is the local slope angle, β the satellite zenith angle, sφ the satellite azimuth angle, and φ the aspect angle of the terrain element.Both ASTER and MODIS LST products were not corrected for terrain angular and adjacency effects. The terrain-induced angular effect can be corrected based on the cosine method  as folloaM is the irradiance from the adjacent terrain and L(x00304) is the local average radiance emitted from the surrounding terrain [In addition to the emittance by the terrain element, the terrain irradiance is also contributed from the adjacent terrain -22. The  terrain . The eff terrain .2.2.i denotes the pixel i at a sub-pixel or fine scale. The upscaled ASTER LST may serve as a basis for investigating the uncertainty embedded in MODIS 1-km LST.ASTER LST has a spatial resolution of 90-m, different from MODIS LST, which are directly incomparable with respect to spatial resolution. It is necessary to upscale ASTER LST before a comparison of ASTER and MODIS data. ASTER LST can be upscaled with a scaling function from a fine resolution into a coarse one. In the case of topographic heterogeneity, the angular and adjacency effects can be accounted for in the follow scaling function :(4)T= and Δε = ε31 − ε32. sT is the retrieved LST (K). T31 and T32 are the brightness temperature (K), and ε31 and ε32 are the emissivity used for MODIS TIR bands 31 and 32, respectively. A2, A3, B2, and B3 are the coefficients in the look-up table defined by Wan and Dozier [a and b can be estimated from multiple regression approach [The MODIS 1-km LST products have been found to be underestimated with the GSW algorithm  over sem follows :(5)Ts′=d Dozier . ɛ′=0.5approach .With the three approaches, we can reduce the discrepancy between ASTER and MODIS LST. Further incorporated with angular and surface features, we may make more insightful analysis into terrain effects on MODIS LST.3.3.1.The study area is located on the Loess Plateau of China. et al. [We used ASTER surface emissivity (AST_05) and surface kinetic temperature (AST_08) products, dated on 8 June 2004. Liu et al. , 12 detaet al. .We acquired the same date MODIS products including MOD02_QKM, MOD02_HKM, MOD02_1KM, MOD03_L1A, and MOD11_L2 data. MOD03_L1A geolocation product provided satellite zenith and azimuth angles for each MODIS 1-km pixel. MOD11_L2 data contains LST and band-averaged emissivity in band-31 (10.780–11.280 μm) and band-32 11.770–12.270 μm) generated using the GSW algorithm. .270 μm g3.2.et al. [MODIS has the geolocation with an accuracy of 18±38 m in-track and 4±40 m cross-track . ASTER het al. ,12 detaiASTER narrowband emissivity was produced from bands 13 and 14 emissivity data. It was used as “accurate emissivity” in LST correction . The broEssentially, MODIS LST is determined from surface emissivity in the GSW algorithm. To evaluate the uncertainty in emissivity, we compared the ASTER and the MODIS emissivity at the corresponding wavelength. To investigate the uncertainties related to surface feature, we generated LST difference images from ASTER LST to original MODIS LST, and to that rectified with or without terrain correction. To find the terrain-induced uncertainty in MODIS LST, we analyzed the relationships of MODIS-to-ASTER LST and emissivity with angular matrix 4.We first report the MODIS-to-ASTER LST discrepancy, which served as a basis for subsequent analysis. With concern on the relationships between MODIS LST and terrain feature, we then analyze the MODIS-to-ASTER discrepancy relevant to angle of local slope and angle of emitted radiance, in addition to spatial distribution of the discrepancy. At last, we address the uncertainty in MODIS emissivity, which directly determines the accuracy of MODIS LST.4.1.p>0.005). The largest difference was at an angle of around 11 degree [The original 1-km MODIS LST was general lower than the ASTER LST. It had a discrepancy to the upscaled ASTER LST with -2.7±1.28K and a root-mean-square-error (RMSE) of 3.02K . The maj1 degree . This de4.2.The original MODIS LST increased generally after rectification with the GSW based approach. Yet, the discrepancy between the rectified LST and the ASTER is as large as 1.2±1.21 K. Even though the S.D. was lower than that of the ASTER-to-original MODIS LST, there existed the relative large differences at the range of 298-305K . These p4.3.Further corrected for terrain effect, the discrepancy was reduced to 0.1±1.33 K on average between ASTER and MODIS LST. Likely, there existed some pixels with a relative large difference at the range of 298-305K . The pix4.4.MODIS emissivity is the key to retrieval of the MODIS 1-km LST. The uncertainty in emissivity directly affects the accuracy of LST. In the present examination, MODIS emissivity in band 31 had a large discrepancy to the corresponding ASTER emissivity and they showed no obvious correlation . As a rep<0.001). Changes in the relationship of emissivity difference versus LST discrepancy supported our inferences on terrain effects [With respect to terrain features, the ASTER-to-MODIS emissivity difference showed no obvious relation with local slope angle . In cont effects .The accuracy of MODIS emissivity relies on classification of land cover . Each cl5.The present study reported the terrain effects on the LST discrepancy between ASTER and MODIS with a case examination over a relief area at the Loess Plateau of China. The direct terrain-induced effect made not the major but was an important influence on the MODIS 1-km LST. A large local slope does not necessary lead to a large LST discrepancy. The angle of emitted radiance was more important than the slope angle in generating the LST discrepancy. Specifically, the conventional terrain correction may be unsuitable for densely vegetated area. The distribution of ASTER-to-MODIS emissivity suggested that the terrain correction was included in the generalized split window (GSW) based approach used to rectify MODIS LST. Further study should include the uncertainty in emissivity, related to classification error, for reliable use of satellite-retrieved LST over relief area.


Nitric oxide (NO) plays a role in thermoregulation and growth of protozoa. This work aimed to add the molecule NO in physiology of protozoa in contact with abused narcotic substances.Paramecia. The cell response to the drug was recorded promptly after drug infusion using a potency protocol provided for the first time at this laboratory. A precursor of NO, L-arginine, was treated jointly with drug to involve the NO system in protozoan performance to drug exposure. Marking of NADPH-diaphorase (NADPH-d) was followed to provide data to explain the mechanisms.A sedative drug, morphine, was infused into a cell chamber containing Paramecia. The infusion of L-arginine (1 to 8 µg/µ1) together with morphine potentiated this effect, though, pre-usage of L-NAME (1 to 8 µg/µ1), a blocker of NO production, reversed the response. Notably the activation of NADPH-d in solely morphine or L-arginine plus morphine samples was revealed. However, the expression of marker was attenuated upon pre-infusion with L-NAME.Morphine particularly (0.5 to 60 µg/µ1) aggregated the Paramecia following exposure to the misused sedative drug, morphine.This study introduces a new approach to involve NO in physiology of aggregation of  Influence of abused drugs in superior animals, the mammals, has been studied extensively , 2. NeurP. caudatum has been explained much more recently by Malvin et al. (2003) (Nitric oxide (NO), a molecule which participates in a variety of physiological functions such as neuronal transmission and cell-cell signaling in the nervous system , blood p. (2003) .Mytilus is defined as the receptor with a high percent (95%) sequence identity to that of human neuronal mu-opiate receptor , an established cytochemical marker of NOS in the living systems . Hay infusions were boiled for about 5 min; the boiled hay was then allowed to settle and the supernatant was used as a culture medium . Each sample (20 ml) of this medium was incubated at 29 to 31°C for about 48 hr.It should be noted that the fresh-water protozoa of the Tehran region have already been classified and recorded by this laboratory .P. caudatum was cultivated in fresh specific medium containing Saccharomyces cerevisiae, a species of budding yeast (2 g/l of sterile distilled water), which was enriched by salts (0.2 g/l CaCl2 and 0.2 g/l MgCl2). This medium was adjusted to a pH of 6.8 ± 0.2 and was maintained at 29 to 31°C.G-Nitro-L-arginine Methyl Ester  were prepared fresh in sterile distilled water (vehicle). The chemical gradients were purchased from Merck Chemicals (Germany).Morphine sulphate , L-arginine  and NP. caudatum was counted daily using a Sedgwick-Rafter cell counting chamber , a useful device for studying the growth of microorganisms. This chamber, holding a little more than 1 ml, can be covered with a thin coverslip, allowing microscopic examination with objectives up to 16X. The cells were counted by light microscopy using a 4X objective. A sample of the cell culture was placed in the chamber, which was then covered with a coverslip. The microorganisms were subsequently allowed to settle in the chamber. This step was performed as quickly as possible to ensure that the protozoa were randomly distributed and settled uniformly in the chamber , the free image processing and analysis program for Microsoft Windows, was used for image analysis after spatial calibrations to provide quantification for an area of 100 µm2.The population of  chamber , 25. TheP. caudatum the experiments were designed and performed as follows:To measure the potency of morphine in Initially, a sample (1 ml of cultivation medium) of the cells was placed in the chamber; permitting cell viability for about 30 min . The celThis phase was started after the pretesting phase, which is also considered step one (the familiarization phase with a length up to 5 sec). This phase was promptly initiated with the infusion of morphine (0.5 to 60 µg/µ1) ; thus, tCell aggregation was measured (counts/view) during the potency phase 60 sec after morphine infusion.After testing, the experimental samples were fixed to provide adequate NADPH staining . The pro2 area was examined in the samples (7 samples) of each experiment by a light microscope (Olympus). Intensity analysis was assessed in 100-µm2 units using the photorecords at 4X magnification with the Image Tool program , the free image processing and analysis program for Microsoft Windows for quantification analysis for an area 100 µm2.Data are presented as mean± SEM. Groups were compared using one-way analysis of variance (ANOVA). Differences between groups were measured by means of Tukey-Kramer post-hoc test. A p-value of < 0.05 was the threshold for statistical significance. The NADPH-diaphorase (NADPH-d) reaction in a 100-µmP. caudatum was cultivated in a specific enriched medium detailed in the Materials and Methods. The number of Paramecia in the medium showed an increase during 48 h ranging from 266 ± 9 to 5999 ± 51 (number of cells/ml). Throughout the ascending phase of the growth phase, a significant increase in the number of Paramecia occurred from 6 hr up to 48 hr  = 49.700, p < 0.0001] over time (sec). The maximum morphine-induced increase in Paramecia numbers occurred at 2 µg/µ1 of the drug. Thus, this dose was used for the subsequent behavioral tests.p < 0.05] effect of L-arginine, the NO precursor, on morphine accumulation potency in P. caudatum. The NO-generating agent potentiated the morphine response in a dose-dependent manner. Based on the results, a dose of 4 µg/µ1 of L-arginine was used for subsequent behavioral testing. Notably, L-arginine itself showed also a significant response . The most effective single dose of the agent was 2 µg/µ1 according to the post-hoc Tukey-Kramer test  = 2118.420, p < 0.0001). The NOS inhibitor blocked the observed effect in a manner independent on the dose. Though, the agent caused no significant response by itself (p > 0.05) ( > 0.05) .p < 0.01) under infusion of L-arginine the NO precursor. The NO agent caused an activation of the NOS, a process leading to NO production and confirming that the NO is participated in the cell aggregation (regation .p < 0.01). The antagonist reversed the effect of the NO precursor L-arginine confirming the NO involvement in accumulation of Paramecia  . A role for L-arginine in NO production has been confirmed in a later study by examining the ability of P. caudatum to produce [3H] L-citrulline from [3H] L-arginine via a mechanism inhibited by L-NAME.Previous data have shown that oduction . Malvin . (2003)  also sugP. caudatum to narcotic drug. This study examined the possible role of the NO system in the unicellular animal P. caudatum in response to the abuse drug morphine. We showed that the aggregation of the cells in contact to morphine was enhanced in the animals pre-infused with the NO precursor, L-arginine. The NO system, therefore, may be involved in morphine-induced aggregation in the single-celled animal P. caudatum.The present results may demonstrate for the first time that the molecule NO is participated in attracting of Thus, this organism may be usable as a new model for measuring of morphine potency in living organisms. Indeed, because that this unicellular model is low-cost and easy to manipulate, it is acquiescent that be used to interrogate the cellular mechanisms governing on opioid dependence.Paramecia, as been previously demonstrated express several NO-sensitive targets, including guanylyl cyclase, potassium channels and voltage gated calcium channels (Stentor have demonstrated that these types of microorganisms express a G protein-mediated response to morphine when stimulated mechanically (The unicellular organisms, channels –32. Otheanically .The NO which has been classified as an important signaling molecule that plays role in various physiological processes in invertebrates  is also P. caudatum, the results of NADPH-d were analyzed that properly support this detail. The fact is that the NO signals the aggregation response to morphine in the single-celled organism, P. caudatum. The NO which has been introduced as a molecule of great pharmacological interest and physiological importance (P. caudatum, the response which is likely mediated by the opioid receptor- and the NO-dependent cell signaling pathways.To quantitatively identify the mechanisms governing on morphine-induced aggregation of portance  participportance . The molportance , 37 and portance . This woP. caudatum was shown by using a novel behavioral assay. This work may properly involve the NO in the functioning of the microorganism to misuse drug exposure.In conclusion, the contribution of molecule NO in morphine-induced aggregation of


Hyperhomocysteinemia is known to cause degeneration of retinal ganglion cells, but its influence on photoreceptors remains largely unknown. In particular, the role of homocysteine-thiolactone (Hcy-T)—the physiologic metabolite of homocysteine that has been proven to be more cytotoxic than homocysteine itself—as a factor that causes retinopathy, has not been defined. This study aimed to investigate the toxic effects of excessive Hcy-T in a mouse model.A total of 60 six-week-old female ICR mice were used in this study. The mice were divided into 3 experimental groups and 2 control groups. The mice in the experimental groups were subjected to intravitreal injections of Hcy-T to reach final estimated intravitreal concentrations at 5, 25, and 200 μM, respectively. Mice without injection (blank) and with 0.9 NaCl injections (sham injection) were used as controls. The mice with 200 μM Hcy-T were sacrificed at days 7, 15, 45, and 90 after injection and the mice with 5 or 25 μM Hcy-T were sacrificed at day 90, with the controls sacrificed at day 15 or 90 for comparison. Semi-quantitative dot-blot analysis was performed for confirmation of retinal homocysteinylation. The mouse retinas were evaluated microscopically, with the thickness of total and specific retinal layers determined. Immunohistochemical analysis was performed and the labeled cells were quantified to determine the effects of excessive Hcy-T on specific retinal cells.+ photoreceptors and the calbindin+ horizontal cells were significantly reduced at day 15, and were nearly ablated at day 90 after 200 μM Hcy-T injection (p<0.001 for both day 15 and day 90), which was not seen in the sham injection controls. The Chx-10+ or the Islet-1+ bipolar cells and the Pax-6+ amacrine cells were severely misarranged at day 90, but no significant reduction was found for both cell types. The GFAP+ Müller cells were activated at day 15, but were not significantly increased at day 90 after the injection.Dose-dependent retinal homocysteinylation after Hcy-T injection was confirmed. The homocysteinylation was localized in the outer and inner segments of photoreceptors and the ganglion cell layer (GCL). Retinal cell degenerations were found in the GCL, inner nuclear layer, and outer nuclear layer at day 90 after 200 µM Hcy-T injection. Significant thickness reduction was found in the total retina, outer nuclear layer, and the outer and inner segment layers. A trend of thickness reduction was also found in the GCL and inner nuclear layer, although this was not statistically significant. The rhodopsinExcessive retinal homocysteinylation by Hcy-T, a condition of hyperhomocysteinemia, could lead to degeneration of photoreceptors, which might lead to retinopathies associated with severe hyperhomocysteinemia or diabetes mellitus.Received: April 1, 2011Accepted: July 14, 2011 Homocysteine (Hcy) is a sulfur-containing amino acid derived from methionine metabolism as an intermediate metabolite. Clinically, the normal range of total plasma Hcy falls between 5 to 15 μmol/l. Patients with Hcy concentrations greater than 15 μmol/l are considered to have hyperhomocysteinemia (hHcy) and the condition is commonly associated with diabetes mellitus ,3. In th−/− retina. As a result of Hcy elevation, distinct alterations were observed in the ganglion, inner plexiform, inner nuclear, and epithelial layers in retinas of cbs−/− and 1-year-old cbs+/− mice. High levels of Hcy were concomitantly located in these retinal layers, particularly the ganglion cell layer (GCL), suggesting direct attack by Hcy [Notably, most hHcy-induced retinopathies are indirectly due to retinal arteriosclerosis and thrombosis. At the cellular level, previous studies have indicated involvement of hHcy in vascular endothelial damage and proliferation of vascular smooth muscle cells ,12. At tk by Hcy ,16. Anotk by Hcy  showed tThe accumulation of Hcy in human cells depends on the activities of methionine synthase, cystathionine β-synthase, and methionyl-tRNA synthetase. Hcy can be metabolized by methionine synthase through its activity to convert Hcy into methionine. Hcy can also be converted into cysteine by cystathionine β-synthase. These two pathways deplete intracellular Hcy in a healthy manner. A third metabolic pathway for Hcy is its conversion into homocysteine -thiolactone (Hcy-T) by methionyl-tRNA synthetase, which occurs under normal physiologic reactions in all human cell types investigated . Both HcAnother interesting, important, and unanswered question is how hHcy can attack retina under in vivo conditions. There has so far been no report on hHcy-induced photoreceptor damage, although an earlier report indicated a non-detectable electroretinogram in a case of combined methylmalonic aciduria and homocystinuria . AnotherIn this study, we conducted intravitreal Hcy-T injections in a mouse model to investigate the direct role of Hcy-T in causing retinopathies. Following the injections, Hcy-T remained detected at day 90 in the retina in a dose-dependent manner, suggesting occurrence of homocysteinylation. The toxic level of intravitreal Hcy-T started at 25 μM in this mouse model. The ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), and the outer segment and inner segment (OS/IS) of photoreceptors were severely affected. With intravitreal Hcy-T level at 200 μM, the ONL were all eradicated at day 90 after the injection. Total retinal thickness was reduced as compared to that of the blank or the sham injection controls. This reduction was also observed in the GCL, INL, ONL, and OS/IS layers, respectively. Immunohistochemical staining localized Hcy-T, mainly in the GCL and OS/IS layers, which persisted at day 90 after the injection. Photoreceptor and horizontal cells were the most affected as indicated by the disappearance of their specific markers. The bipolar, amacrine, and Müller glial cells were all disorganized. Our results indicate that excessive Hcy-T leads to more extensive retinal homocysteinylation than hHcy and most retinal cells, including photoreceptors, are damaged under the influence of excessive Hcy-T.A total of 60 six-week-old female ICR mice were used in this study. The mice were purchased from National Laboratory Animal Center . The mice weighed about 25 g on arrival and were fed ad libitum and kept under standard conditions with a 12 h:12 h light-dark cycle. The mice were acclimatized and habituated to the laboratory for at least one week before the experiments. All animal maintenance and experiment protocols were reviewed and approved by the Animal Care and Use Committee of Chung Shan Medical University and were performed in agreement with the Association for Research in Vision and Ophthalmology (ARVO) Resolution on the Use of Animals in Research.Prior to intravitreal injection, the mice were anesthetized with intraperitoneal injections of sodium pentobarbital (45 mg/kg bodyweight). Intravitreal injection procedures and criteria were applied following the details published by Moore . BrieflyDot-blot analysis was used to test the levels of retinal homocysteinylation after intravitreal Hcy-T injections. After the mice were euthanatized, their eyes were extracted, followed by removal of the cornea and the lens tissues and separation of the retinas from the eye cups. The retinas were homogenized in radioimmunoprecipitation (RIPA) buffer  containing a protease inhibitor cocktail and were then centrifuged at 10,000× g for 15 min at 4 °C. The supernatants were collected and assayed for their protein levels with a Bio-Rad Protein Assay kit  using BSA as standard. Equal aliquots (30 μg) of the retinal proteins were applied to a 96-well dot blot apparatus (Life Science Research) and were then transferred to a nitrocellulose membrane by vacuum filtration. The preparation was immersed in 10% non-fat milk for 30 min at room temperature and was then incubated with the rabbit anti-homocysteine antibody  overnight at 4 °C, followed by incubation with the peroxidase-linked antirabbit IgG antibody  for 1 h at room temperature. After thorough washing, chemiluminescence was developed  and detected with a digital imaging system . Intensities of dots stained with anti-homocysteine were determined by calibration of the total amounts of pixels using the ImageJ image processing program . The retinal homocysteinylation levels in the injection groups were represented as the relative amounts of pixels compared with those of the retinas without injection (blank controls). Six independent samples from each injection group were determined. To ensure independent observation, only one eye from each mouse was randomly selected for assessment of retinal homocysteinylation.For microscopic evaluations, the mice were sacrificed by cervical dislocation. One eye from each mouse was randomly selected and extracted. The extracted eyes were fixed in 4% formalin for at least 24 h, washed with 0.9% NaCl, and processed through ethanol and xylene solutions. The preparations were then embedded in paraffin, cut at 5-μm thickness, and mounted on glass slides following conventional procedures –28. Hema); mouse anti-calbindin ; mouse anti-Pax-6 antibody ; rabbit anti-Islet-1 ; sheep anti-Chx-10 ; or rabbit anti-GFAP . The preparations were then incubated with a horseradish peroxidase-conjugated secondary antibody (1/200), either antimouse, antirabbit, or antisheep IgG . After incubation, the preparations were washed thoroughly, incubated in diaminobenzidine tetrahydrochloride solution for color detection, and were counterstained with hematoxylin. Positively labeled cells were counted on two sites, approximately 200 to 300 μm, apart on either side of the optic nerve. The areas for counting were fixed at 100 μm in width for each site. The Rhodopsin positive cells were counted in the ONL. The calbindin positive cells were counted in the OPL. The Islet-1 or Chx-10 or Pax-6 positive cells were counted in the INL. The glial fibrillary acidic protein (GFAP) positive cells were counted in the whole thickness of the retina. The results of counting were then averaged for that particular section. For each retina analyzed, three separate sections were measured by two observers without prior knowledge of the study groups. All quantifications of labeled cells were performed with a Nikon E100 microscope (Nikon) equipped with a digital camera and the ImageJ image processing program .For immunohistochemical analysis, the retinal tissue sections were boiled in a citrate buffer (pH 6.0) for 20 min for antigen retrieval and were then incubated, respectively, with one of the following antibodies: rabbit anti-homocysteine ; mouse anti-rhodopsin  and were compared among groups. The retinal thickness and the means of labeled retinal cells were compared and analyzed by the Mann–Whitney test. All statistical analyses were performed using the Prism program .We first tried to determine whether intravitreal Hcy-T injection can lead to homocysteinylation in the retina. Dot-blot analysis was performed using the supernatant of the whole retina at day 90 after the injection. Retinal homocysteinylation was found in a dose-dependent manner, with the highest extent of homocysteinylation found in the retinas injected with Hcy-T to reach a final concentration of 200 μM . QuantitWith retinal homocysteinylation confirmed after Hcy-T injections for final concentrations of 5, 25, and 200 μM, we then examined the effects after this manipulation. There was no evidence of the degeneration of specific retinal layers following Hcy-T injection for 5 μM. Hcy-T injection for 25 μM, however, started to result in retinal cell degeneration, particularly in the GCL, INL, and ONL, where most of the retinal cell nuclei are located . FurtherTo further understand the effects of excessive Hcy-T on the retina, we compared the localization of retinal homocysteinylation by immunohistochemistry at days 7, 15, 45, and 90 after injections for a final intravitreal Hcy-T concentration at 200 μM. Generally, the sites of retinal homocysteinylation were most evident in the OS/IS and the GCL layers , as comp+ photoreceptor cells were significantly reduced , horizontal , bipolar (Chx-10 or Islet-1), amacrine (Pax-6), and Müller cells (GFAP) at day 15 and day 90 following the injections. At day 15, the rhodopsin reduced  as compa reduced . This re reduced , which w reduced . The calnjection  and werenjection , in contnjection  and day njection , respectnjection . At day njection , which wnjection . The samnjection , in contnjection . The Paxnjection , but thenjection , as compnjection  and day njection . The GFAnjection , which wnjection . Howevernjection , as compnjection .+ photoreceptor cells and calbindin+ horizontal cells were both significantly reduced . Ruction of cell populations was also observed in the Chx-10+  has been implicated in human cardiovascular and neurodegenerative diseases, particularly under diabetic conditions ,3,16. In+ photoreceptors in the ONL after Hcy-T injection for 200 μM, as compared to the age-matched 0.9% NaCl sham injection controls  in the OPL was also found after Hcy-T injection for 200 μM as compared to the age-matched 0.9% NaCl sham injection controls (+ (+ (+ cells) and bipolar cells after Hcy-T injection seemed to differ from that of the OS/IS photoreceptor layers. Hcy-T might directly target the OS/IS layers since homocysteinylation was localized there. In contrast, the disruption of the horizontal cells and bipolar cells appeared to be due to indirect effects, as no apparent homocysteinylation was localized within these cells. Functionally, bipolar and horizontal cells act as interneurons to transfer information from photoreceptors to ganglion cells [Apart from photoreceptor degeneration, significant loss of horizontal cells (calbindincontrols . Ructiontrols (+  or the Ils (+ (+  bipolar on cells . Reduction cells –48. Theron cells ,24,25, i+ Müller cells were significantly activated at day 15 after intravitreal Hcy-T injections, but the activation was not seen at day 90, as no significant increase in GFAP+ Müller cells was detected (+ Müller cells at day 15 may represent a functional response to deplete the damaged retinal cells after Hcy-T injection. This early effort obviously failed as the GFAP+ Müller cell population was reduced to nearly the same level as that in the sham-injection control at day 90. The result from this mouse model indicates the risk of chronic hyperhomocysteinemia in a period of just 90 days.Interestingly, the GFAPdetected . Müller We conclude that excessive Hcy-T could cause degeneration of photoreceptors in the mouse retina. Our results support the necessity of evaluating photoreceptors and the OS/IS junction in patients with severe hyperhomocysteinemia or diabetes mellitus.